This application claims priority to German Application No. 100 28692.5 filed Jun. 9, 2000.
The invention concerns a process for examining of membrane enclosed biocompartments, wherein the biocompartments are arranged in amicro-flow chamber. This micro-flow chamber is subject to the through-flow of a culture medium containing an active agent and which is in contact with the said biocompartments and possibly has an effect thereon. Further, the pH value of the culture medium present in the micro-flow chamber can be measured. In this arrangement, the biocompartments can hold, for instance, biological cells, microorganisms (fungi, bacteria), biochemical compartments and/or mitochondria.
EP 0 394 406 B1 discloses a process of the type known in the art, in which living, biological cells are placed in a micro-flow chamber being subjected to the through-flow of a culture medium and which chamber has a silicon sensor on its bottom. A reference electrode is provided in the micro-flow chamber, which is in continual contact with the culture medium. In the case of this previously known process, in a first process step, the through-flow of the culture medium is stopped in the micro-flow chamber. Under these circumstances, the pH value, serving as a metabolic indicator of substance change in the culture medium residing in the micro-flow chamber, is determined by measurement of the electrical potential between the silicon-sensor and a reference electrode. With this process, it is possible to measure, for instance, the action of a cell-affecting agent on the metabolism of the cells. A detriment, however, lies in the fact, that the process enables only one determination in regard to a yield of acid metabolic products, while other changes caused by the metabolism of the cells are not given consideration.
DE 44 17078 A1 makes known a process wherein biological cells in a micro-flow chamber are simultaneously examined by means of a plurality of different micro-sensors. As this is carried out, in addition to the acidification, at the same time the respiration of the cells in the culture medium is monitored, which enables a more exact investigation of the action on the cells of an agent contained in the culture medium. This procedure, however, has the disadvantage, that the measured values for pH and oxygen content are determined at different places and thus of differing biological cells present in the micro-flow chamber. Cell cultures, cell divisions, tissues and the like which are to undergo examination as biological material are, however, often heterogenous, that is, different cells can yield different signals. In this known procedure, on this account, imprecisions in the investigation of an agent in the culture medium can occur if the pH value and/or the oxygen content at the points of determination of the sensors deviate in pH and oxygen content from one another.
Thus, it is the object of the invention, to make available a process for the examination of membrane enclosed biocompartments, which enables the measurement at one location of several metabolic parameters of the said biocompartments.
The achievement of this object is found especially in that:
a) the concentration of substance which is yielded or absorbed by metabolism in the micro-flow chamber in a partial increment of the culture medium in the active area of the biocompartments is indirectly measured, since between an operational electrode placed in said partial increment and a distanced reference electrode, an electrical potential is so applied to the culture medium, that hydroxyl or hydrogen ions are formed from substances in the culture medium,
b) at a measuring point in the partial increment of the culture medium during the application of the said electrical voltage, at least one first value for a pH value of the culture medium is determined,
c) subsequently, the electrical voltage will be turned off or changed in such a way, that the formation of hydroxyl ions and hydrogen ions from the said substance ceases,
d) shortly before or after the measurement of the first value at shut-off voltage, or by a voltage in which the generation of hydroxyl and hydrogen ions from the said substance is not possible, at least a second measurement value is determined for the pH value of the culture medium,
e) from the first and the second measured values, a difference is calculated,
f) the steps a) to e) are repeated at least once,
g) from a difference between at least two of these measured value differences, the concentration change of the material in the culture medium, and from the difference between at least two of the first pH measurements or the second pH values, the acidification or alkalinizations of the culture medium can be determined,
h) and from the so obtained measurement values for the concentration change as well as the acidification or alkalinization, the metabolic activity of the biocompartments can be determined.
The basis of the process lies in the recognition, that the biocompartments continually change the pH value of the culture medium at a somewhat constant increase. Further, this alteration of pH value runs essentially slower than the pH change arising from an application of electrical voltage for the generation of hydroxyl ions (OHxe2x88x92 ions) or hydrogen ions (H+ ions) from substances in the culture medium yielded by, or absorbed by metabolism in the biocompartments. This contributes to the fact that it becomes possible in spite of the superimposing of the ions of hydroxyl and/or hydrogen yielded to or taken from the culture medium by the biocompartments with those ions of hydroxyl or hydrogen from substance carried in the culture medium, at one and the same location, and with only a single pH-sensor, to measure both the substance which arises from the concentration change activated by the biocompartments of the material yielded to or taken from the biocompartments as well as the pH change in the culture medium caused by the biocompartments. With the inventive process, it becomes possible to determine the respiration of the biocompartments by the measurement of the oxygen concentration change, as well as the concentration change of nitrogen monoxide (NO), hydrogen peroxide, and/or other compounds containing oxygen and/or hydrogen in a chemically bound form, such as, for instance, molecules which are present in the cell surfaces. A dependency of change of concentration and/or pH value based on localization caused by a difference in the individual cells of the cell culture has practically no effect on the accuracy of the determination of the metabolism of the cells undergoing examination.
From Sohn, B.-H., Kim, C.-S., xe2x80x9cA new pH-ISFET-based dissolved oxygen sensor by employing electrolysis of oxygenxe2x80x9d in Sensors and Actuators B34, (1996), pages 435-440, is, to be sure, an already disclosed process, contrary to the generic type, in which the oxygen content in analytes is measured indirectly by means of the pH value, wherein the electrical potential on the analyte is so far reduced in the negative direction, that from such oxygen as is in the analyte, hydroxyl ions are formed, and wherein these hydroxyl ions are detected by means of an ion selective sensor. The analyte contains, however, no biocompartments which change during the measurement, that is, which change the analyte by means of acid metabolic products and the consumption of oxygen. Also, this known procedure results in no evaluation of the sensor signal in regard to the determination of respirational activity and acidification when potential is not reduced.
In a particularly advantageous embodiment of the process, a provision is made that the sequence comprising the steps a) to g) is run through at least twice and that in these at least duplicated operations, the polarity is differently chosen between the electrical potential applied between the operational electrode and the reference electrode. This makes it possible to measure in the culture medium, using only one pH-value sensor, both the concentration change of electro-negative substances, such as oxygen, as well as the concentration of electro-positive substances, such as nitrogen monoxide or hydrogen peroxide.
With this arrangement, in the measurement of the change in concentration of an electro-negative substance, there is applied to the operational electrode, an electrical potential which is negative relative to the potential of the reference potential. Conversely, if the operational electrode is to measure the concentration change of an electro-positive substance, then the applied potential is to be, in the same manner, positive.
It is advantageous, if the time for the measurement of the pH-value is arranged to fall in such a manner on the time at which the electrical potential changes or is shut off, so that the ion concentrations of respectively hydrogen and hydroxyl at the measurement instant are essentially in equilibrium. The metabolism, that is, the viability of the biocompartments can be measured with greater precision with such timing.
In another advantageous embodiment of the invention, provision has been made, that respectively, the period, between the moment of the application, the shutting off, or changing of the electrical voltage, and the subsequent time at which a pH measurement is made, is essentially made the same for all pH values. In this way, in the forming of a difference between the first and second pH values upon the measurement of the concentration change in the culture medium by the material involved in the metabolic activity of the biocompartments that is, from a first pH value from the measurement of the change of pH value in the culture medium caused by the biocompartments, compensation is provided for what effect time may have in regard to the period between the beginning of a potential change and the subsequent measurement of the pH value. This contributes to an even greater accuracy in measurement.
It is of particular advantage, if the electrical voltage is cut off, or, in accord with step c) is changed, before a state of equilibrium has established itself in the hydrogen ion and the hydroxyl ion concentrations in the culture medium. The biocompartments in the micro-flow chamber are then only subjected for a short time to the application of the electrical potential required for the formation of the hydroxyl ions or hydrogen ions. Thus, the biocompartments remain substantially unaffected in their physiological condition by the application of potential.
This situation enables the carrying out of measurements on the biocompartments for a longer period. This provides the ability to successively bring more different agents in contact with the biocompartments and to compare their various activities on the biocompartments with one another.
In an advantageous embodiment of the process, the electrical current flowing between the operational electrode and the reference electrode is measured. The curve of the electrode current can then be compared with the curve of the electrical voltage applied to the electrodes, and if necessary, compared also with a previously stored Amp/Volt-characteristic line or with a family of such curves, which will provide another checkpoint in monitoring the plausibility of the results of the measurements.
Expediently, the biocompartments are so placed in reference to the operational electrode, that theyxe2x80x94at least in an area sensexe2x80x94cover over the electrodes. From the time related curves of the electrical potential between the operational electrode and the reference electrode, along with that of the electrode current, a possible alteration of the morphology or adhesion of the biocompartments can be determined.
It is of particular advantage, if the pH value is measured by means of an Ion Selective Field Effect Transistor (hereinafter ISFET). Because of the capability of such a sensor to respond in a range of small signals, the inventive process is enabled to make a high degree of placement determination, whereby it is even possible to investigate single cells of the cell culture and to compare the thereby obtained measurements with each other. In this manner, for instance, in a cell culture, single cells which react especially sensitively to a given agent can be localized.
In another advantageous embodiment of the invention, the through-flow of the culture medium is stopped, or at least reduced, during and in between the taking of the first and last pH measured values of a provided series of pH value determinations for the establishing of the respiratory activity of the biocompartments and the pH value change activated by said biocompartments in the culture medium. The oxygen content diminishes during the time following the stopping or reducing of the culture medium through-flow in a case of oxygen consuming biocompartments.
Conversely, in the case of photosynthetic biocompartments which generate oxygen, the oxygen content will increase. In this situation, however, a correspondingly large measurement signal is produced, which makes possible an exact determination of the respiratory activity of the biocompartments. Where biocompartments are concerned which consume oxygen, after the stopping or reducing of the culture medium through-flow, an increase in the pH value of the culture medium occurs, and in the case of biocompartments, which produce oxygen, a decline in pH values is evident. In this way, there is available a correspondingly large measurement signal because of the pH value change of the culture medium activated by the biocompartments.